ORIGINAL RESEARCH
AFFILIATIONS 1Marmara Üniversitesi. Eczacılık Fakültesi, Farmasötik Kimya, İstanbul, Türkiye 2Marmara Üniversitesi. Eczacılık Fakültesi, Farmasötik Biyoteknoloji, Istanbul, Türkiye CORRESPONDENCE Sevim Rollas E-mail: srollas@marmara.edu.tr Received: 28.11.2011 Revision: 13.12.2011 Accepted: 15.12 .2011 INTRODUCTION
The use of thiosemicarbazide in organic synthesis has become a classical strategy for the synthesis of several heterocycles. Among the increasing number of heterocyclic sulphur and nitrogen containing compounds, which are being pursued in both industry and academia, 1,3,4-thiadiazole and 1,2,4-triazole derivatives are also interesting targets for drug design. Therefore, there have been intense investigations on 1,4-disubstituted-thiosemicarbazide, 1,3,4-thiadiazole and 1,2,4-tri-azole-thione compounds.
1,4-Disubstituted-thiosemicarbazide are biologi-cally versatile compounds displaying a variety of biological effects which include anti-inflammato-ry (1), antimycobacterial (2, 3), antimicrobial (4-6), antifungal (7), antibacterial (8, 9) and antiviral (3) activities. 2,5-Disubstituted 1,3,4-thiadiazoles,
synthesis of which frequently include the reac-tion of acylthiosemicarbazides with acidic rea-gents such as concentrated sulfuric acid, possess various biological properties such as anticonvul-sant (10), antifungal (11, 12), antituberculosis (13-15), antimicrobial (16, 17), anti-inflammatory (18), cytotoxic (19), and antiproliferative (20), antioxidant (21-22) activities. In addition, 1,2,4-triazole-thiones which possess important pharmacological activities such as anticonvul-sant (23), anti-inflammatory (24-28), antibacterial (29), cytotoxic (30, 31), antimicrobial (32), anti-cancer (33,34), and antiviral (35) have found wide use in medicinal chemistry as common struc-tures.
The present communication deals with the syn-thesis of 1-(α,α-diphenyl-α-hydroxy) acetyl-4-substitutedthiosemicarbazide (2a-k),
[5-(substi-ABSTRACT: This study describes the synthesis of novel 1-(,-diphenyl--hydroxy)acetyl-4-substitutedthiosemicarbazide (2a-k), [5-(substitutedamino)-1,3,4-thiadiazole-2-yl](diphenyl) methanol (3a-b) and 3-[hydroxy(diphenyl)methyl]-4-(nonsusbtituted/substituted)-2,4-dihy-dro-5H-1,2,4-triazole-5-thione derivatives (4a-c) and evaluation of their cytotoxic activities. In the course of the syntheses benzilic acid methyl ester was reacted with hydrazine hydrate in absolute ethanol to afford benzilic acid hydrazide (1). Reaction of 1 with appropriate alkyl/ arylisothiocyanates gave 1-(,-diphenyl--hydroxy)acetyl-4-substitutedthiosemicarbazide (2a-k). [5-(substitutedamino)-1,3,4-thiadiazole-2-yl](diphenyl)methanol derivatives (3a-b) were obtained by cyclization of 2a and 2c with concentrated sulphuric acid. On the other hand, 3-[hydroxy(diphenyl)methyl]-4-(nonsusbtituted/substituted)-2,4-dihydro-5H-1,2,4-tria-zole-5-thione (4a-c) were obtained by cyclization of 2c, 2d and 2g with 2N NaOH. The struc-tures of the new compounds were confirmed by the data obtained from elemental analysis, HPLC, UV, IR, 1H-NMR, 13C-NMR, HSQC and MS spectra. Compounds 2a, 2c-k, 3b and 4b were selected for cytotoxic screening by using HEK293 cell line of MTT assay. The highest inhibition were confirmed as 50.23% at 10 mg/ml for the compound 1-(,-diphenyl--hydroxy)acetyl-4-cyclohexylmethylthiosemicarbazide (2e).
KEY WORDS: thiosemicarbazide, thiadiazole, 1,2,4-triazole-5-thione, cytotoxic activity.
Synthesis and evaluation of cytotoxic
activities of some 1,4-disubstituted
thiosemicarbazides,
2,5-disubstituted-1,3,4-thiadiazoles and 1,2,4-triazole-5-thiones
derived from benzilic acid hydrazide
tutedamino) -1,3,4-thiadiazole-2-yl] (diphenyl) methanol
(3a-b) and 3-[hydroxy (diphenyl) methyl]-4-(nonsusbtituted/
substituted)-2,4-dihydro-5H-1,2,4-triazole-5-thione deriva-tives (4a-c). Their structures were confirmed by means of UV, IR, 1H-NMR, Mass spectral data and elemental analysis. All the synthesized compounds were screened for their cytotoxic activities by using HEK293 cell line of MTT assay.
EXPERIMENTAL Chemistry
All solvents and chemicals used in this study were supplied from Aldrich, Merck and Fluka and used without purification. Melting points (ºC) were measured using Schmelzpunktbes-timmer SMP II melting point apparatus, uncorrected. The re-actions were monitored on Merck pre-coated aluminium TLC plates 60F–254 and the products were visualized by UV-light using ethyl asetate and ether (50:50, v/v) as solvent system. The UV spectra were recorded on a Schimadzu UV-1601 spec-trophotometer. The Infrared spectra were recorded on Schi-madzu FTIR-8400S Spectrophotometer and expressed in wave number ν (cm-1). 1H-NMR spectra were recorded on Bruker AVANCE-DPX 400 and Varian Mercury Spectrometer at 400 MHz, using DMSO-d6 as a solvent; tetramethylsilane (TMS) was used as internal standard. All NMR chemical shifts are reported as values in parts per million (ppm) and coupling constant (J) are given in hertz (Hz). Mass spectra were ob-tained by using Agilent 1100 LC-MS and Waters 2695 Alliance Micromass. 1H-NMR and Mass analyses were provided by Faculty of Pharmacy Center Laboratory (Ankara University) and the Scientific and Technical Research Council of Turkey, TÜBİTAK (Ankara).
Preparation of benzilic acid hydrazide (1)
Hydrazine hydrate (0.03 mol, 80%) was added to benzilic acid
methyl ester (0.01mol). The mixture was refluxed at 100C for
30 minutes. After adding ethanol (10.0 mL), the mixture was heated in a water steam bath for two hour. The residue was filtered, washed with water and recrystallized from ethanol. mp: 171–172 C, (lit. [36] mp. 169-170 C).
General procedure for the synthesis of 1-(α,α-diphenyl-α-hydroxy)acetyl-4-(substituted)thiosemicarbazide (2a-k)
Equimolar amounts of benzilic acid hydrazide (0.01 mol) and appropriate alkyl/aryl isothiocyanates were refluxed in etha-nol (30 mL) for 2-3 hours. The crystalline product was filtered and recrystallized from ethanol to obtain 1,4-disubstituted-thiosemicarbazides.
1-(α,α-diphenyl-α-hydroxy)acetyl-4-(2-chloroethyl)thiosem-icarbazide (2a): White crystals, yield 86%, mp 260–262 oC; IR (νmax, cm-1): 3248 (NH), 1707 (C=O), 1244 (C=S). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 3.51 (-CH2Cl, protons are shadowed with solvent), 3.97 (2H, t, NHCH2), 7.08 (1H, s, OH), 7.21–7.63 (10H, m, Ar-H), 10.80 (1H, N4-H), 11.09 (1H, s, N2-H), 12.00 (1H, s, N1-H). Anal. Calcd for C
17H18ClN3O2S (363.86): C, 56.12; H, 4.99; N, 11.55; S, 8.81%. Found: C, 56.23; H, 4.85; N, 11.48; S, 8.99%. (API-ES+, m/z, %): 328 [M+-35.5] (100), 310, 282, 210, 209, 208, 178, 150, 143.
1-(α,α-diphenyl-α-hydroxy)acetyl-4-(4-cyanophenyl)thiose-micarbazide (2b): White crystals, yield 85%, mp 169–173 oC; IR (νmax, cm-1): 3363 and 3300 (NH), 1687 (C=O), 1282 (C=S). 1H
NMR (400 MHz, DMSO-d6) δ (ppm): 6.82 (1H, s, OH), 7.24– 7.78 (14H, m, Ar-H), 9.34 (1H, N2-H), 10.12 (1H, s, N1-H), 10.50 (1H, s, N4-H). Anal. Calcd for C22H18N4O2S (402.46): C, 65.65; H, 4.51; N, 13.92; S, 7.97%. Found: C, 65.33; H, 4.34; N, 14.08; S, 8.38%. (API-ES+, m/z, %): 403 [M+], 388, 386, 385 (100), 379, 366, 355, 347, 342, 307, 301, 294, 268, 264, 254, 246.
1-(α,α-diphenyl-α-hydroxy)acetyl-4-benzoylthiosemicarbazi-de (2c): White crystals, yield 42%, mp 189–190 oC; IR (νmax, cm -1): 3232 (NH), 1674 (C=O), 1251 (C=S). 1H NMR (400 MHz, DM-SO-d6) δ (ppm): 7.28 (1H, s, OH), 7.13–8.04 (15H, m, Ar-H), 10.86 (1H, N4-H), 11.93 (1H, s, N2-H), 13.17 (1H, s, N1-H). Anal. Calcd for C22H19N3O3S (405.46): C, 65.17; H, 4.72; N, 10.36; S, 7.91%. Found: C, 64.91; H, 4.60; N, 10.36; S, 7.16%. (API-ES+, m/z, %): 407 (M++H)+, 389 (100), 388, 338, 276, 247, 229, 199, 157.
1-(α,α-diphenyl-α-hydroxy)acetyl-4-(4-trifluorometho-xyphenyl)thiosemicarbazide (2d): White crystals, yield 48%,
mp 145–146 oC; IR (νmax, cm-1): 3300 and 3142 (NH), 1653 (C=O), 1261 (C=S). 1H NMR (400 MHz, DMSO-d
6) δ (ppm): 6.78 (1H, s, OH), 7.65–7.14 (12H, m, Ar-H), 9.28 (1H, N4-H), 9.97 (1H, s, N2-H), 10.50 (1H, s, N1-H). Anal. Calcd for C
22H18 F-3N3O3S (461.45): C, 57.26; H, 3.93; N, 9.11; S, 6.95%. Found: C, 57.37; H, 4.00; N, 9.08; S, 6.63%. (API-CI+, m/z, %): 462 [M+], 446, 445, 444 (100), 252, 225, 223, 208, 79.
1-(α,α-diphenyl-α-hydroxy)acetyl-4-cyclohexylmethylthio-semicarbazide (2e): White crystals, yield 94%, mp 194–196 oC; IR (νmax, cm-1): 3162 (NH), 1687 (C=O), 1294 (C=S). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 0.65–1.84 (11H, m,cyclohexyl), 3.28 (2H, m, –CH2), 6.76 (1H, s, OH), 7.09–7.59 (11H, m, Ar-H and N4-H), 9.34 (1H, s, N2-H), 10.26 (1H, s, N1-H). Anal. Calcd for C22H27N3O2S (397.53): C, 66.47; H, 6.85; N, 10.57; S, 8.07%. Found: C, 66.32; H, 6.84; N, 10.51; S, 8.04%. (API-ES+, m/z, %): 398 [M+], 381, 342, 304, 292, 264, 263, 225, 208, 188, 173.
1-(α,α-diphenyl-α-hydroxy)acetyl-4-(3-pyridyl)thiosemicar-bazide (2f): White crystals, yield 85%, mp 184–186 oC; IR (ν
max, cm-1): 3182 (NH), 1651 (C=O), 1257 (C=S). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 6.75 (1H, s, OH), 7.17–7.38 ve 7.45–7.60 (10H, m, Ar-H), 7.40 (1H, dd, J=8.07 Hz, 8.08 Hz, pyridine-H5), 7.93 (1H, d, pyridine-H4), 8.36 (1H, d, pyridine-H6), 8.53 (1H, s, pyridine-H2) 9.33 (1H, N4-H), 10.02 (1H, s, N2-H), 10.56 (1H, s, N1-H). Anal. Calcd for C20H18N4O2S (378.44): C, 63.47; H, 4.79; N, 14.80; S, 8.47%. Found: C, 63.26; H, 4.83; N, 14.58; S, 8.15%. (API-CI+, m/z, %): 379 [M+] (100), 243, 226, 225, 197, 139, 138, 137.
1-(α,α-diphenyl-α-hydroxy)acetyl-4-(3,5-bistrifluoromethyl-phenyl)thiosemicarbazide (2g): White crystals, yield 29%, mp
166–168 oC; IR (ν
max, cm-1): 3252 and 3186 (NH), 1651 (C=O), 1273 (C=S). 1H NMR (400 MHz, DMSO-d
6) δ (ppm): 6.79 (1H, s, OH), 7.22–7.75 (10H, m, Ar-H), 7.88 (1H, s, Ar-H4), 8.26 (2H, s, Ar-H2, Ar-H6), 9.64 (1H, s, N2-H), 10.27 (1H, s, N1-H), 10.55 (1H, N4-H). Anal. Calcd for C
23H17F6N3O2S (513.45): C, 53.80; H, 3.34; N, 8.18; S, 6.24%. Found: C, 54.44; H, 3.39; N, 8.29; S, 6.24%. (APCI-, m/z, %): 512 (M+-H)+, 511 (100), 286, 252.
1-(α,α-diphenyl-α-hydroxy)acetyl-4-(2-chloro-5-trifluorome-thylphenyl)thiosemicarbazide (2h): White crystals, yield 58%,
mp 152–154 oC; IR (ν
max, cm-1): 3302 and 3180 (NH), 1693 (C=O), 1261 (C=S). 1H NMR (400 MHz, DMSO-d
6) δ (ppm): 6.65 (1H, s, OH), 7.27–7.86 (13H, m, Ar-H), 10.75 (1H, N4-H),
9.06 (1H, s, N2-H), 10.09 (1H, s, N1-H). Anal. Calcd for C22H17ClF3N3O2S (479.90): C, 55.06; H, 3.57; N, 8.76; S, 6.68%. Found: C, 55.21; H, 3.58; N, 8.72; S, 6.82%.
1-(α,α-diphenyl-α-hydroxy)acetyl-4-(2-trifluoromethyl-phenyl)thiosemicarbazide (2i): White crystals, yield 64%, mp
168–170 oC; IR (ν
max, cm-1): 3271 and 3194 (NH), 1651 (C=O), 1278 (C=S). 1H NMR (400 MHz, DMSO-d
6) δ (ppm): 6.64 (1H, s, OH), 7.14–7.82 (14H, m, Ar-H), 9.02 (1H, N4-H), 9.94 (1H, s, N2-H), 10.55 (1H, s, N1-H). Anal. Calcd for C
22H18F3N3O2S (445.45): C, 59.32; H, 4.07; N, 9.43; S, 7.20%. Found: C, 58.36; H, 4.51; N, 8.73; S, 6.80%. (API-ES+, m/z, %): 446 [M+] (100), 428, 368, 302, 296, 279, 257, 225, 192.
1-(α,α-diphenyl-α-hydroxy)acetyl-4-(2-methylsulphanyl-phenyl)thiosemicarbazide (2j): White crystals, yield 63%, mp
164–166 oC; IR (ν
max, cm-1): 3325 and 3261 (NH), 1651 (C=O), 1255 (C=S). 1H NMR (400 MHz, DMSO-d
6) δ (ppm): 2.34 (3H, s, S-CH3), 6.72 (1H, s, OH), 7.11–7.60 (14H, m, Ar-H), 9.00 (1H, N4-H), 9.82 (1H, s, N2-H), 10.53 (1H, s, N1-H). Anal. Calcd for C22H21N3O2S2 (423.55): C, 62.39; H, 5.00; N, 9.92; S, 15.14%. Found: C, 62.21; H, 4.93; N, 9.89; S, 14.92%.
1-(α,α-diphenyl-α-hydroxy)acetyl-4-(1-adamantyl)thiosemi-carbazide (2k): White crystals, yield 83%, mp 160–162 oC; IR (νmax, cm-1): 3313 and 3180 (NH), 1651 (C=O), 1282 (C=S). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 1.06 (3H, t,–CH3 of etha-nol), 1,48–2.59 (16H, m, adamantyl-H), 3.40–3.53 (2H, m,–CH2 of ethanol), 4.36 (1H, t, OH of ethanol), 6.12 (1H, N4-H), 6.92 (1H, s, OH), 7.23–7.48 (10H, m, Ar-H), 9.13 (1H, s, N2-H), 10.26 (1H, s, N1-H). 13C NMR (100 MHz, DMSO-d 6) δ (ppm): 19.21, 29.64, 36.58, 39.51, 39.72, 39.93, 40.14, 40.35, 40.55, 40.77, 41.54 (adamantyl), 53.68 (solvent), 56.76 (NH-adamantyl carbon), 81.05 (C-OH), 128.10, 128.32 (Ar), 144.21 (C=O), 180.00 (C=S). Anal. Calcd for C25H29N3O2S.C2H5OH (481.65): C, 67.33; H, 7.32; N, 8.72; S, 6.66%. Found: C, 67.14; H, 7.07; N, 8.79; S, 6.51%. (API-CI+, m/z, %): 436 [M+] (100), 418, 226, 225, 243.
General procedure for the synthesis of [5-(substitutedamino)-1,3,4-thiadiazole-2-yl](diphenyl)methanol (3a-b)
A sample of corresponding 1,4-disubstituted-thiosemicar-bazides (0.001 mol) was treated with concentrated sulphuric acid at room temperature with constant stirring for 1-2 h. The reaction mixture was poured into ice water. The product was precipitated, filtered and washed with water to afford 3a-b in quantitative yield.
[5-(ethenylamino)-1,3,4-thiadiazole-2-yl](diphenyl)methanol (3a): White powder, yield 81%, mp 202–203 oC; IR (νmax, cm-1): 3257 (NH), 1614 (C=N). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 3.66 (t, 2H, =CH2, J= 6.86 Hz, 13.77 Hz), 4.76 (s, 1H, OH), 6.79 (d, 1H, -CH=, J=7.70 Hz), 6.93–7.42 (m, 10 H, ArH), 7.81 ve 8.18 (2s, 1H, NH). Anal. Calcd for C17H15N3OS (309.38): C, 66.00; H, 4.89; N, 13.58; S, 10.36%. Found: C, 65.12; H, 4.86; N, 13.11; S, 10.13%. (API-ES+, m/z, %): 310 [M+] (100), 256, 208, 192.
[5-(benzoylamino)-1,3,4-thiadiazole-2-yl](diphenyl)methanol (3b): Yellow crystals, yield 87%, mp 206–208 oC; IR (ν
max, cm-1): 3250 and 3165 (NH), 1600 (C=N). 1H NMR (400 MHz, DMSO-d
6) δ (ppm): 7.04–8.17 (m,16 H, ArH ve OH), 12.44 (s, 1H, -NHCO). Anal. Calcd for C22H17N3O2S (387.45): C, 68.20; H, 4.42; N, 10.85; S, 8.28%. Found: C, 68.20; H, 4.61; N, 10.91; S, 8.90%.
General procedure for the synthesis of 3-[hydroxy(diphenyl) methyl]-4-(nonsusbtituted/substituted)-2,4-dihydro-5H-1,2,4-triazole-5-thione (4a-c)
Sodium hydroxide (2N, 15-20 mL) was added to correspond-ing 1,4-disubstituted-thiosemicarbazides and refluxed on wa-ter bath for 4h. The reaction mixture was neutralized with hy-drochloric acid (10%). The precipitate was filtered, washed with water and crystallized from ethanol.
3-[hydroxy(diphenyl)methyl]-2,4-dihydro-5H-1,2,4-triazole-5-thione (4a): White crystals, yield 49%, mp 258 oC; IR (ν
max, cm-1): 3107 (NH), 1577, 1487 (C=N, NH), 1260 (C-N), 1180 (C=S), 1H NMR (400 MHz, DMSO-d6) δ (ppm): 6.95 (1H, s, OH), 7.11–7.58 (10H, m, Ar-H), 13.20 (1H, s, NH), 13.40 (1H, s, NH). Anal. Calcd for C15H13N3OS.1/2 H2O (292.35): C, 61.62; H, 4.83; N, 14.37; S, 10.97%. Found: C, 60.97; H, 4.60; N, 13.84; S, 10.64%. 3-[hydroxy(diphenyl)methyl]-4-[4-(trifluoromethoxy) phenyl]-2,4-dihydro-5H-1,2,4-triazole-5-thione (4b): White crystals, yield 84%, mp 235–237 oC; IR (ν max, cm-1): 3169 (NH), 1510, 1558 (C=N, NH), 1271 (C-N), 1155 (C=S). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 6.91 (2H, d, Ar-H, J= 8.9 Hz), 7.00 (1H, s, OH), 7.15 (2H, d, Ar-H, J= 8.2 Hz), 7.18–7.35 (10H, m, Ar-H), 13.49 (1H, s, NH). Anal. Calcd for C22H16F3N3O2S.H2O (461.46): C, 57.26; H, 3.93; N, 9.11; S, 6.95%. Found: C, 58.04; H, 4.03; N, 9.05; S, 6.34%. (API-ES+, m/z, %): 444 [M+], 443, 426, 425, 388, 314, 261, 79. 4-[3,5-bis(trifluoromethyl)phenyl)]-3-[hydroxy(dipenyl) methyl]-2,4-dihydro-5H-1,2,4-triazole-5-thione (4c): White crystals, yield 78%, mp 215–216 oC; IR (ν max, cm-1): 3365 (NH), 1556, 1492 (C=N, NH),1276 (C-N), 1120 (C=S). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 7.12 (1H, s, OH), 7.18–7.34 (10H, m, Ar-H), 7.52 (2H, s, triazole ArC2-H, ArC6-H), 8.07 (1H, s, tria-zole ArC4-H), 14.14 (1H, s, NH). Anal. Calcd for C23H15F6N3OS (495.44): C, 55.76; H, 3.05; N, 8.48; S, 6.47%. Found: C, 55.86; H, 2.86; N, 8.55; S, 6.19%.
Cytotoxic Activity
The synthesized compounds were tested for their cytotoxic ac-tivities. Cell viability and cytotoxic activity profile of the com-pounds were analyzed using the Cell Proliferation Kit I (MTT) [Roche, Germany]. MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide] is cleaved to formazan crystals by the “succinate-tetrazolium reductase” system which be-longs to the mitochondrial respiratory chain and is active only in viable cells (37-38). HEK293 cell line was used for the deter-mination of cytotoxic activity. The MTT metabolic assay was carried out in 96-well flat-bottom cell culture plates seeded with 5x103 cells/well. HEK293 cells in 100mM containing L-Glutamine without antibiotic Eagle’s MEM (Minimum Essen-tial Medium) and RPMI 1640MEM with 10% FBS (Fetal Bovine Serum).
The following day, media was aspirated and the compounds were solved in DMSO and diluted with medium before they were added to the cell cultures at the concentrations of 5.0 g/ mL and 10.0 g/mL. Cells were incubated for 48 hrs at 37ºC, 5.0% CO2. After the incubation period add 10 L of the MTT labeling reagent (final concentration 0.5 mg/mL) to each well. Incubate the microplate for 4-12 hrs in a humidified
atmos-phere (e.g. 37oC, 5.0% CO
2) and add 100 L of the solubiliza-tion solusolubiliza-tion into each well. Allow the plate to stand overnight in the incubator in a humidified atmosphere (e.g. 37oC, 5.0% CO2), the formazan crystals solubilized. Absorbance of forma-zan product was measured with spectrophotometrically at 550 and 690 nm.
RESULTS AND DISCUSSION
In this research, 1-(α,α-diphenyl-α-hydroxy)acetyl-4-substitut-edthiosemicarbazide (2a-k),
[5-(substitutedamino)-1,3,4-thia-diazole-2-yl](diphenyl)methanol (3a-b) and
3-[hydroxy(diphenyl)methyl]-4-(nonsusbtituted/
substituted)-2,4-dihydro-5H-1,2,4-triazole-5-thione deriva-tives (4a-c) were synthesized. In the first part of the study, ben-zilic acid methyl ester was reacted with hydrazine hydrate in absolute ethanol to afford benzilic acid hydrazide (1). Reaction of 1 with appropriate alkyl/arylisothiocyanates gave 1-(α,α-diphenyl-α-hydroxy)acetyl-4-(substituted)thiosemicarbazide
(2a-k). [5-(Substitutedamino)-1,3,4-thiadiazole-2-yl](diphenyl)
methanol derivatives (3a-b) were obtained by cyclization of 2a and 2c with concentrated sulphuric acid. On the other hand, 3-[hydroxy(diphenyl)methyl]-4-(nonsusbtituted/
substituted)-2,4-dihydro-5H-1,2,4-triazole-5-thione (4a-c) were obtained by cyclization of 2c, 2d and 2g with NaOH (2N)
(Scheme 1). During the cyclization of 2a, 2-chloroethyl group
converted to ethenyl group in the acidic media. Unexpectedly,
4a was obtained from the cyclization reaction of 2c by missing
the benzoyl group. Ergenç et. al. (39) reported different meth-odology for the synthesis of the compound 4a therefore it was necessary to elucidate the structure of this compound.
The preference formation of the 1,3,4-thiadiazole ring under acidic condition can be due to the loss of nucleophilicity of N-4 as a result of its protonation leading to a comparable increase in the nucleophilicity of the sulphur atom towards the attack of the carbonyl carbon. On the other hand, when cyclization
Scheme 1. Reactions and conditions; (i) hydrazine hydrate; (ii) (Ar)R-N=C=S; (iii) 2N NaOH, 10% HCl;
was carried out under alkaline conditions, the nucleophilicity of N-4 was enhanced and led to cyclization the carbonyl car-bon atom to give 1,2,4-triazole-5-thiones. Because of the alka-line media, the nitrogen atom of acylthiosemicarbazides is more nucleophilic than either the oxygen of the carbonyl group or the sulphur of the thiocarbonyl group. The reaction mechanism of 1,3,4-thiadiazole derivatives (3a-b) is shown in Scheme 2 and a possible mechanism for the synthesis of 1,2,4-triazole-5-thiones (4a-c) is shown in Scheme 3 (40,41). All the synthesized compounds have been characterized by means of both analytical and spectroscopic methods. The IR spectra of the 1,4-disubstituted thiosemicarbazide derivatives
(2a-k) have C=O stretching bands at 1651–1707 cm–1 and C=S bands at 1244–1294 cm–1. The 1H-NMR spectra of compounds
2a-k showed signals at 10.09–13.17, 9.06–11.93 and 6.12–10.86
ppm, attributed to N1H, N2H and N4H-Ar(R), respectively (42). On the other hand, 13C-NMR spectra of the compound 2k exhibited resonance at 180.00 and 144.21 ppm assigned for C=S and C=O moieties, respectively. For compounds 4a-c the IR spectra showed bands around 1120–1180 cm-1 characteris-tics for C=S bending vibrations and around 1260–1276 cm-1 characteristics for C-N vibrations, providing evidence for ring closure. In the IR spectra of compounds 3a-b and 4a-c no ab-sorption bands were detected about 1651–1707 cm–1 indicating the absenceC=O group of compounds 2a-k which is an evi-dence for the conversion of thiosemicarbazides to thiadiazoles and triazoles. From cyclization of compounds 2a-k under alka-line conditions, only the thione type compounds 4a-c were ob-served by the presence of absorption maxima at 1120–1180 cm–1 belonging to C=S group. (43). The 1H-NMR (DMSO-d
6) spectrum of compound 3b displayed NH resonance in 12.44 ppm (44). Further structural confirmation was provided by the HSQC spectrum of compound 3a which showed the expected 13C-1H correlations. (M+-H)- ions with 100% abundance
ob-served in the atmospheric pressure chemical ionization [APCI] was spectra of compound 2g, [M+] ions observed in [API-ES+]
Scheme 2. Proposed mechanism for the synthesis of 2,5-disubstituted-1,3,4-thiadiazoles.
Figure 1. Absorbance values of after MTT assay formazan
crystalls of 1,4-disubstituted thiosemicarbazides.
and [API-CI+] were spectra of compounds 2a-f, 2i, 2k, 3a, 4b provided further confirmation for the formation of the expect-ed structures. Mass spectra of these compounds gave molecu-lar ion peaks except compound 2a, however, with different intensities. Because of the loss of H2O and isothiocyanate mol-ecules, the major fragmentation pathway in the 1,4-disustitut-ed thiosemicarbazide derivatives formation of C14H11NO2 + (m/z 225) ion is observed. Besides, some compounds undergo specific fragmentations.
Cytotoxicity of the selected compounds were evaluated by us-ing HEK293 cell line accordus-ing to procedures of MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bro-mide] assay. All the compounds were dissolved in dimethyl-sulfoxide (2.5%) and no cytotoxic effect on the cells was ob-served when compared to the control group. 5.0 g/mL and 10.0 g/mL were used in two different doses and dose-de-pendent cytotoxic activity was investigated. Cell viability and cytotoxic activity profile of the compounds were analyzed us-ing the MTT assay.
Cytotoxic activity results were presented in Figures 1-4. The cytotoxic activity range of 1,4-disubstituted-thiosemicar-bazides is 0–30.72% at 5.0 g/mL concentration. Even these values are below 50% for the 5.0 μg/mL concentrations, we could not mention about cytotoxic compounds. The cytotoxic activity range is 9.59–50.23% at 10.0 g/mL concentration. Based on the gained data 4 compounds which belong to our set of ten 1,4-disubstituted thiosemicarbazide derivatives demonstrated inhibition between 5–20%, the other four com-pounds 20–30%, whereas the other two comcom-pounds demon-strated inhibition between 40–50% (Figure 1). Only one com-pound which have cytotoxicity with the value of 50.23% at 10.0 g/mL is 1-(α,α-diphenyl-α-hydroxy)acetyl-4-cyclohexyl-methylthiosemicarbazide (2e). We have also made comparison of cyctotoxic activity of thiosemicarbazides with their cyclic derivatives, thiadiazoles and triazoles (Figure 3). [5-(Benzoylamino)-1,3,4-thiadiazole-2-yl](diphenyl)methanol (3b), which is derivative of compound 2c and 3-[hydroxy(diphenyl)methyl]-4-[4-(trifluoromethoxy)
Scheme 3. Proposed mechanism for the synthesis of 1,2,4-triazole-5-thiones.
Figure 3. Absorbance values of after MTT assay formazan
crystalls of thiadiazole and triazoles.
phenyl]-2,4-dihydro-5H-1,2,4-triazole-5-thione (4b), which is derivative of 2d have not showed any cytotoxic activity at the dose of 5.0 g/mL. As statistically significant results, the cyto-toxic activity of the three heterocyclic compounds that were used in the synthesis, increases depending on the dose (p<0.05), which means they showed a dose-related effect at these concentrations.
CONCLUSIONS
A series of 1,4-disubstituted-thiosemicarbazides, 2,5-disubsti-tuted-1,3,4-thiadiazoles and 1,2,4-triazole-5-thiones were
syn-thesized and screened for their cytotoxic activities against HEK293 cell line. The cytotoxicity screening indicated that among the tested compounds 1-(α,α-diphenyl-α-hydroxy) acetyl-4-cyclohexylmethylthiosemicarbazide (2e) exhibited cytotoxic activity.
ACKNOWLEDGEMENTS
This study was financially supported by the Marmara Univer-sity Scientific Research Committee (Project No: SAG-C-YLP-090909-0286).
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Benzilik asid hidrazidinden elde edilen bazı 1,4-disübstitüe tiyosemikarbazidler,
2,5-disübstitüe-1,3,4-tiyadiazoller ve 1,2,4-triazol-5-tiyonların sentezi ve sitotoksik aktivitelerinin değerlendirilmesi
ÖZET
Bu çalışmada 1-(,-difenil--hidroksi)asetil-4-sübstitüetiyosemikarbazid (2a-k), [5-(sübstitüeamino) -1,3,4-tiyadiazol-2-il] (difenil)metanol (3a-b) ve 3-[hidroksi(difenil)metil]-4-(nonsübstitüe/sübstitüe)-2,4-dihidro-5H-1,2,4-triazol-5-tiyon (4a-c) yapısındaki yeni bileşikler sentez edilmiş ve bileşiklerin sitotoksik etkinlikleri araştırılmıştır. Bu amaçla benzilik asit metil esterinin hidrazin hidrat ile etanollü ortamda reaksiyonu ile benzilik asit hidrazidi (1) elde edilmiş, 1’in uygun alkil/aril isotiyosiyanatlara katımı ile 1-(,-difenil--hidroksi)asetil-4-sübstitüetiyosemikarbazid (2a-k) ka-zanılmıştır. [5-(sübstitüeamino)-1,3,4-tiyadiazol-2-il](difenil)metanol türevi (3a-b) bileşikler, 2a ve 2c’ nin derişik sül-fürik asit ile siklizasyonu sonucu elde edilmiştir. 3-[hidroksi(difenil)metil]-4-(nonsübstitüe/sübstitüe)-2,4-dihidro-5H-1,2,4-triazol-5-tiyon türevi (4a-c) bileşikler ise 2c, 2d ve 2g’nin 2N NaOH ile reaksiyonundan kazanılmıştır. Bileşiklerin yapıları elemental analiz, HPLC, UV, IR, 1H-NMR, 13C-NMR, HSQC ve MS verileri ile doğrulanmıştır. Bileşik 2a, 2c-k, 3b ve 4b HEK293 hücre hattı kullanılarak MTT yöntemiyle sitotoksik etkinlikleri araştırmak üzere seçilmiştir. Özellikle 1-(,-difenil--hidroksi)asetil-4-siklohekzilmetiltiyosemikarbazid (2e) bileşiğinde 10 mg/ml konsantrasyonda % 50.23 sitotoksik aktivite saptanmıştır.
ANAHTAR KELİMELER: tiyosemikarbazid, tiyadiazol, 1,2,4-triazol-5-tiyon, sitotoksik aktivite
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